The long-term goal of this project is to investigate the fundamental interactions among oxygen, nitric oxide (NO) and hemoglobin (Hb) at the level of the microcirculation, using combined experimental and theoretical approaches. Nitric oxide can affect two key aspects of oxygen supply and demand (1) through its action as a vascular smooth muscle relaxant and (2) through its action as an inhibitor of cytochrome c oxidase. The ability of enzymatic sources of NO to account for experimental values of [NO] in and around microvessels will be assessed by analyzing immunohistochemical localization of NOS isoforms, applying specific inhibitors of NOS to experimental animals, measuring [NO] using microelectrodes and a new fluorescence technique, and comparing the results with predictions of specialized biochemical models of NO production, where specific NOS isoforms can be removed from the calculation (in silico knockout). Experiments will be carried out on the spinotrapezius muscle of normotensive (WKY) and spontaneously hypertensive rats (SHR). The balance between oxygen supply and demand will be altered by changing one or both variables. Oxygen supply will be altered by changing systemic hematocrit (normovolemic hemodilution or hemoconcentration) and oxygen demand will be increased by electrically stimulating the spinotrapezius muscle. Plasma and tissue PO2 will be measured using phosphorescence quenching microscopy, hemoglobin oxygen saturation in microvessels will be measured microspectrophotometrically, microvascular geometry and hemodynamic parameters will be measured using video imaging techniques, oxygen consumption will be measured by the stop-flow technique, and [NO] will be measured as described above. The microvascular responses to reduced oxygen supply to demand ratio will be related to changes in [NO]. The degree to which NO changes in response to the decreased oxygen supply/oxygen demand will be compared between the WKY and SHR, and the experimental results will be compared with predictions of the computational model that employs parameters specific to the WKY and SHR subjects. We will also theoretically analyze the pathways that lead to NO synthesis in the microvasculature and their contributions to regulating oxygen delivery and demand. An integrative model that will combine the biochemical pathway analysis of NO production, the biotransport of NO, and the distribution of oxygen will be developed. The experimentally determined vascular and tissue PO2 and [NO] will be thoroughly compared with the predictions of the biophysically and biochemically detailed computational models. Thus, the project will include state-of-the-art combined experimental and computational studies that should lead to a better quantitative understanding of NO release and dynamics and the interplay between NO release and oxygen delivery, two cornerstones of vascular biology. Public Health Relevance: The long-term objective of the project is to provide a physiological basis for understanding the mechanisms of nitric oxide (NO) production, as well as to gain a better knowledge of the general mechanisms of NO transport in the vasculature. Moreover, the proposed studies will provide information that will lead to better treatment of various cardiovascular diseases through utilizing NO-induced vasodilation and oxygen delivery pathways.